A new endogenous signaling molecule named Calexcitin has been identified recently in Hermissenda. Calexcitin was first identified by a change in phosphorylation of a protein band in neurons that were classically conditioned in a comparative study. Calexcitin is a Ca++ and GTP binding protein, and has a PKC site. Calexcitin interacts with RyR to induce Ca release from the ER, however when it is phosphorylated it moves to the plasma membrane were it causes inactivation of potassium channels. Calexitin was not yet shown to exist in mammalian cells and its role in Ca++ metabolism in non-excitable cells was not studied. Our project at the NIH BioCurrent Center aimed to characterize the response of potassium currents and calcium fluxes to calexcitin in mammalian lymphocytes. We took two approaches, one which is recording the acute response to exposure to purified squid Calexcitin applied in lyposomes while the other was to record the response to over expression of transfected squid calexcit in. Calexcitin was packed into lyposomes and applied on lymphocyte while Ca concentration was monitored using fluo -3. We could record a calcium elevation that lasted 40 second. This effect was repeatable and did not appear when Calexcitin B was used. To characterize the source of the calcium, we are now repeating the experiment in the presence of no extracellular Ca or while recording Ca fluxes using a Ca selective self-referencing electrode. To learn about the effect of long lasting intracellular calexcitin we made a cDNA construct of squid calexcitin fused to enhanced GFP or BFP (Blue Fluorescent Protein) at its carboxy terminal. These were transfected into immortalized lymphocytes in a plasmid vector containing a CMV promoter and neomycin resistance. Transient and stable expression were studied using both fluorescence and confocal microscopy. Calexitin was found localized to the plasma membrane as well as to the nucleus. The pattern of localization following various stimuli is currently being studied. Transfected cells were subjected to patch clamp recording, in whole cell configuration and potassium currents were recorded. The basal Ca concentration of transfected cells as well as their response to intracellular Ca elevation and depletion is currently being studied.